JP7364798B2 - capillary electrophoresis device - Google Patents

Description

The present invention relates to technology for capillary electrophoresis devices.

Biopharmaceuticals have superior effects that small-molecule drugs do not have, in that antibody molecules modified with sugar chains are effective against specific targets such as cancer and rare and intractable diseases. While small-molecule drugs are synthesized through chemical reactions, biopharmaceuticals are produced using the biological functions of cells, so slight changes in culture conditions can affect the molecular structure of the product. Immunoglobulin G (IgG), a typical biopharmaceutical, is a large molecule with a complex structure and a molecular weight of about 150,000. It is nearly impossible to prevent structural heterogeneity in such large molecules. Therefore, quality inspection technology plays an even more important role in biopharmaceuticals to confirm the safety and effectiveness of formulations. Since the target substances such as immunoglobulin G have complex structures, there are a wide variety of test items for biopharmaceuticals. Capillary electrophoresis is used as a test item for biopharmaceuticals, such as confirmation tests to confirm that the main components contained in the test object are target substances, and purity tests to evaluate the content of impurities. In a capillary electrophoresis device, a sample such as an antibody is injected into a capillary and electrophoresed, whereby the sample is separated according to its molecular weight and charge amount, and detected by a detection section installed near the end of the capillary. . Optical methods such as ultraviolet (UV) absorption, native fluorescence (NF), and laser induced fluorescence (LIF) are widely used as detection methods.

Patent Document 1 states, "A sample detection device 1 is provided with disk substrates 1a and 1b through which a light beam a passes, and is obtained by bonding the substrates 1a and 1b together. A flow path 4 for conducting polymers is formed in the sample detection device 1. In the sample detection device 1, a dielectric material for reflecting the light beam a is provided on the back side of the surface on which the light beam a is incident, and in a region corresponding to the region where the flow path 4 is formed. According to the sample detection device 1 in which the first reflective film 12 consisting of The present invention discloses a sample detection device and a sample detection apparatus that can be used for both systems, and the size of the main body of the sample detection apparatus can be easily reduced.

Japanese Patent Application Publication No. 2005-147954

Capillary electrophoresis devices use very thin capillaries with an inner diameter of 5 to 250 μm in order to suppress the generation of Joule heat. Therefore, the area that is irradiated with the measurement light, which is the light for measuring the capillary, is very small. Since the area irradiated with the measurement light is very small, there is a problem in that the detection sensitivity is low (the minimum detectable sample concentration is large). Furthermore, electrophoresis of one sample requires a long time (typically about 30 minutes). Therefore, low throughput is also an issue. In Patent Document 1, optical measurement is performed while rotating a disk substrate provided with four electrophoresis channels while irradiated with light (measurement light), and the throughput is increased by making it possible to measure four samples at the same time. is improving. However, in such a structure, most of the time the measurement light is irradiated onto areas other than the flow path. Therefore, the irradiation time of measurement light per channel becomes short, and the sensitivity decreases. Furthermore, since the flow path is created on a disk substrate that can be rotated at high speed, the length of the flow path is shortened, resulting in a problem that electrophoretic separation performance is reduced.

The present invention was made in view of this background, and an object of the present invention is to provide a highly sensitive capillary electrophoresis device.

In order to solve the above problems, the present invention includes a light source section, an irradiation optical section that transmits light emitted from the light source section back and forth to a capillary, and an optical signal based on the light transmitted back and forth to the capillary. and a first light detection unit for detecting, and the irradiation optical unit detects a first polarization component, which is one of the polarization components of the light irradiated from the light source unit, in order from the light source unit side. a polarization separation unit that transmits the light of the polarized light component and reflects the light of the second polarized light component that is the other polarized light component; a λ/4 plate provided in the front stage of the capillary; The device is characterized in that it includes a reflecting portion that reflects the light of the first polarized component toward the capillary .
Other solutions will be described as appropriate in the embodiments.

According to the present invention, a highly sensitive capillary electrophoresis device can be provided.

FIG. 1 is a schematic diagram showing an example of the overall configuration of a capillary electrophoresis device according to a first embodiment. It is a figure showing an example of composition of a detection device in a 1st embodiment. 1 is a diagram illustrating a configuration example of an analysis device used in a first embodiment. FIG. FIG. 2 is a schematic diagram showing an example of the overall configuration of a capillary electrophoresis device according to a second embodiment. It is a figure showing an example of composition of a detection device in a 2nd embodiment. This is an example (in the case of 7 fiber bundles) showing a cut surface at an arbitrary location of the fiber bundle. This is an example (in the case of 8 fiber bundles) showing a cut surface at an arbitrary location of the fiber bundle. It is a graph of simulation results showing the relationship between the position of the optical fiber in the fiber bundle and the amount of light. It is a graph showing the absolute value of the fluctuating amount of light that occurs when the input side end face position of the optical fiber fluctuates by Δr in the r direction. It is a figure showing an example of composition of a detection device in a 3rd embodiment. It is a figure showing an example of composition of an analysis device used in a 3rd embodiment. It is a graph showing a change in the light condensing position of a lens due to control of a galvanometer mirror.

Next, modes for carrying out the present invention (referred to as "embodiments") will be described in detail with reference to the drawings as appropriate. In addition, in each drawing, the same reference numerals are given to the same components, and the description thereof will be omitted.

[First embodiment]
(Overall configuration diagram of capillary electrophoresis device E)
FIG. 1 is a schematic diagram showing an example of the overall configuration of a capillary electrophoresis device E according to the first embodiment.
The capillary electrophoresis device E includes a detection device 1, an analysis device 2, a capillary 5, a high voltage power source 8, and an electrode 9.
As shown in FIG. 1, the electrophoresis medium container 6 and the sample container 3 contain a sample and an electrophoresis medium, respectively. Before the measurement, the capillary 5 is first connected to the electrophoresis medium container 6. Then, the electrophoretic medium is injected into the capillary 5 by electrical means, pressure injection, or the like. When the capillary 5 is sufficiently filled with the electrophoresis medium, the capillary 5 is connected to the sample container 3. Then, the sample is injected into the capillary 5 by electrical means, pressure injection, or the like.

The injection-side electrode tank 4 and the discharge-side electrode tank 7 are each filled with a buffer solution. During measurement, the end of the capillary 5 and the end of the electrode 9 are immersed in the injection-side electrode tank 4 and the discharge-side electrode tank 7, respectively. When voltage is applied to each of the injection side electrode tank 4 and the discharge side electrode tank 7 by the high voltage power supply 8, each molecule of the sample is separated by electrophoresis according to properties such as molecular weight and charge amount. The capillary 5 is moved from the injection side 151 toward the discharge side 152. When each molecule of each sample that has moved reaches the detection device 1, it is detected in the detection device 1 by optical means. Further, an analysis device 2 is connected to the detection device 1 . The analyzer 2 receives a signal from the detector 1 and performs the analysis necessary to analyze the molecules in the sample based on the signal. Note that the capillary electrophoresis device E includes a high-voltage power supply 8 and a control device (not shown) that controls the pressure for injecting the electrophoresis medium and sample into the capillary 5. Note that the capillary 5 has a tubular quartz tube whose periphery is coated with polyimide. However, inside the detection device 1, the polyimide of the capillary 5 is peeled off, leaving the quartz tube exposed.

(Configuration of detection device 1)
FIG. 2 is a diagram showing a configuration example of the detection device 1 in the first embodiment.
The detection device 1 includes, in order from the light source 101 side, a light source 101, a lens 102, a bandpass filter 103, a polarizing beam splitter 104, a λ/4 plate 107, a lens 108, a lens 109, a lens 110, and a mirror 111. A capillary 5 is disposed between the lens 108 and the lens 109 and at the condensing position of the lens 108 and the lens 109. Further, a reference condensing lens 105 and a reference photodiode 106 are provided on one of the paths of light reflected from the polarizing beam splitter 104. Furthermore, a measuring condensing lens 112 and a measuring photodiode 113 are provided on the other side of the path of the light reflected from the polarizing beam splitter 104.

In FIG. 2, solid line arrows indicate the outward path of light, and dashed line arrows indicate the return path of light.
First, light emitted from the light source 101 (light source light R1) is converted into collimated light by the lens 102. Here, the lens 102 is a collimating lens (first collimating lens). Thereafter, only light of a specific wavelength is extracted by bandpass filter 103. When measuring UV absorption of proteins, the transmission wavelength of the bandpass filter 103 is 280 nm, 220 nm, or the like. Moreover, when performing autofluorescence (Native Fluorescence; NF) measurement, the transmission wavelength of the bandpass filter 103 is 280 nm or the like. Furthermore, when performing laser induced fluorescence (LIF) measurement, the transmission wavelength of the bandpass filter 103 is set to the excitation wavelength of the fluorescent dye to be used. When the emission spectrum of the light source 101 includes only the target wavelength component, the bandpass filter 103 can be omitted. Below, a case where UV absorption measurement is performed will be explained.

The light source light R1 that has passed through the bandpass filter 103 is separated by the polarizing beam splitter 104 into a reference light R2 having an S polarization component (light having a second polarization component) and a measurement light R3 having a P polarization component (light having a first polarization component). (solid line arrow indicated by symbol Y1). Here, the reflective surface 104A of the polarizing beam splitter 104 reflects the S-polarized light component, but transmits the P-polarized light component. The reflective surface 104A is inclined with respect to the traveling direction of the collimated light converted by the lens 102 so that the S-polarized light component reflected toward the reference photodiode 106 (second light detection unit) is directed. installed in the condition. Therefore, the S-polarized light component is reflected toward the reference photodiode 106. Here, the light reflected by the polarizing beam splitter 104 is referred to as reference light R2, and the light transmitted through the polarizing beam splitter 104 is referred to as measuring light R3.

The reference light R2 reflected by the polarizing beam splitter 104 toward the reference photodiode 106 is focused by the reference condensing lens 105 and detected by the reference photodiode 106. Here, the splitting ratio between the reference light R2 and the measurement light R3 by the polarizing beam splitter 104 is 1:1 when a light source 101 that emits non-polarized light, such as a lamp, is used. Furthermore, when a light source 101 that emits polarized light, such as a laser light source, is used, the branching ratio differs depending on the polarization state. However, in such a case, for example, by installing a λ/2 plate after the bandpass filter 103, the polarization state can be adjusted and the branching ratio can be freely set. The following description will be made on the assumption that a lamp light source is used as the light source 101.

On the other hand, the measurement light R3 transmitted through the polarizing beam splitter 104 is converted into circularly polarized light by the λ/4 plate 107, and then focused by the lens 108 and irradiated onto the capillary 5. Here, the lens 108 is a collimating lens (second collimating lens). The measurement light R3 transmitted through the capillary 5 is converted into collimated light by the lens 109, which is a collimating lens. Then, the measurement light R3 converted into collimated light by the lens 109 is focused on the mirror 111 by the lens 110 and irradiated thereon. Note that the lens 110 is a collimating lens (third collimating lens). The measurement light R3 reflected by the mirror 111 follows the same optical path in the opposite direction and is again focused and irradiated onto the capillary 5. The measurement light R3 transmitted through the capillary 5 is converted into collimated light by the lens 108, which is a collimating lens. The measurement light R3, which has been converted into collimated light by the lens 108, has its polarization state changed from circularly polarized light to S-polarized light by the λ/4 plate 107.

As described above, the polarizing beam splitter 104 has the property of reflecting S-polarized light. Therefore, the measurement light R3 converted into S-polarized light is completely reflected by the polarization beam splitter 104 (dashed arrow indicated by the symbol Y2). At this time, the measurement light R3 is reflected toward the measurement photodiode 113 (first photodetector) due to the inclination direction of the reflective surface 104A in the polarizing beam splitter 104. The measurement light R3 reflected in the direction of the measurement photodiode 113 is focused by the measurement condensing lens 112 and detected by the measurement photodiode 113.

Note that in this embodiment, by monitoring the intensity of the reference light R2 with the reference photodiode 106, it is possible to suppress the influence of fluctuations in the intensity of the source light R1 emitted by the light source 101 and improve detection sensitivity. It is.
Alternatively, the signals detected by the reference photodiode 106 and the measurement photodiode 113 are input to the analysis device 2. The analyzer 2 can also divide the signal detected by the measurement photodiode 113 by the signal detected by the reference photodiode 106, and display the result.

Note that the reference condensing lens 105 and the reference photodiode 106 can be omitted.

(Analyzer 2)
FIG. 3 is a diagram showing an example of the configuration of the analyzer 2 used in the first embodiment.
The analysis device 2 includes a CPU (Central Processing Unit) 201, a storage device 202 such as an HD (Hard Disc), a communication device 203, an output device 204 such as a display, and a memory 210.
Here, the communication device receives signals from the detection device 1 and sends control signals to the detection device 1 as necessary.
Further, a program is loaded into the memory 210 from the storage device 202, and the loaded program is executed by the CPU 201. As a result, the calculation section 211 and the analysis section 212 are realized.
The calculation unit 211 performs calculation processing such as dividing the signal detected by the measurement photodiode 113 by the signal detected by the reference photodiode 106.
The analysis unit 212 analyzes the components of the sample based on the results of the calculation unit 211, the state of the signal detected by the reference photodiode 106, the state of the signal detected by the measurement photodiode 113, etc. . The analysis unit 212 then causes the output device 204 to output the analysis results.

(Effect of improved sensitivity)
In the first embodiment, the capillary 5 is irradiated twice with the light (measurement light R3) emitted from the light source 101. With such a configuration, the optical path length of the measurement light R3 irradiated onto the sample in the capillary 5 is doubled, making it possible to improve detection sensitivity. That is, by irradiating the sample in the capillary 5 with the measurement light R3 twice, the light attenuation due to the sample is doubled. Thereby, SNR (Signal to Noise Ratio) can be improved. In other words, sensitivity can be improved.

In the first embodiment, by arranging the lenses 108, 109, 110 and the mirror 111 in this order from the light source 101 side, the capillary 5 is irradiated with the measurement light R3 twice in a round trip. Furthermore, by using the polarizing beam splitter 104 and the λ/4 plate 107 in combination, it is possible to detect the entire amount of the measurement light R3 that is irradiated to the capillary 5 in a reciprocating manner.

This effect will be explained in detail below using a formula.
In UV absorption measurement, absorbance A defined by the following equation (1) serves as an evaluation index.

Here, _I0 is a detection signal detected by the measurement photodiode 113 at a reference time. The reference time is the time when no sample is yet present in the area of the capillary 5 that is irradiated with the measurement light R3. For example, the time when electrophoresis is started is set as the reference time. Moreover, I is a detection signal at an arbitrary time. Note that the detection signal is proportional to the amount of light incident on the measurement photodiode 113. When a sample is injected into the capillary 5 by electrophoresis, the sample passes through a region of the capillary 5 that is irradiated with the measurement light R3. Then, the measurement light R3 is absorbed by the molecules of the sample and the detection signal I decreases, so that an absorbance peak appears in the electropherogram. Based on Beer-Lambert's law, the magnitude S of absorbance by molecules of a sample is expressed by the following equation (2).

S=εcL...(2)

Here, ε is the molar extinction coefficient of the sample molecules, c is the molar concentration of the medium, and L is the optical path length of the medium (mixture of electrophoresis medium and sample) through which the measurement light R3 passes, which is mainly determined by the inner diameter of the capillary 5. be.
On the other hand, the absorbance signal noise N is given by the following equation (3) using the detection signal noise ΔI.

Equation (3) is approximately expressed by the following equation (4) by expanding in the first order of ΔI/I ₀ .

The detection signal noise ΔI is composed of three components: light intensity noise proportional to the amount of light, shot noise proportional to the square root of the amount of light, and detector noise. Detector noise is noise originating from the measurement photodiode 113. The detection signal noise ΔI can be expressed as the following equation (5) using the amount of light P incident on the measurement photodiode 113.

Here, a, b, and c are constants that characterize the magnitude of light intensity noise, shot noise, and detector noise, respectively. Since I ₀ is proportional to P, the absorbance noise N can be expressed as the following equation (6) from equations (4) and (5).

Note that in equation (6), the proportionality constant applied to the entire equation is omitted for simplification. From equations (2) and (6), the SNR of the signal (optical signal) from the measurement light R3 received by the measurement photodiode 113 is given by the following equation (7).

The SNR shown by equation (7) is the SNR of the conventional electrophoresis apparatus in which the capillary 5 is irradiated with the measurement light R3 only once. In this embodiment, the light intensity is reduced by half by the polarizing beam splitter 104 (P in equation (7) becomes P/2), and the optical path length is doubled due to the round-trip irradiation. (L in equation (7) becomes 2L). Considering these, the SNR1 of the measurement light R3 received by the measurement photodiode 113 in the capillary electrophoresis device E of this embodiment can be expressed as the following equation (8).

Comparing equations (7) and (8), in the capillary electrophoresis device E of this embodiment, the light intensity noise component a is 1/2 times higher and the light shot noise component is 1/√2 times higher than before. It can be seen that this has effectively become smaller. Therefore, SNR1>SNR holds true. In other words, the capillary electrophoresis device E according to the first embodiment can achieve higher detection sensitivity than ever before.

In the first embodiment, the case where UV absorption measurement is performed has been described, but it is also applicable to NF measurement and LIF measurement. When applied to NF measurement or LIF measurement, for example, it is possible to insert a long-pass filter or the like that blocks excitation wavelength components and transmits fluorescence wavelengths in front of the measurement condensing lens 112.

Further, in the first embodiment, the reference light R2 split by the polarizing beam splitter 104 on the outward path is detected by the reference photodiode 106. Then, the calculation unit 211 of the analyzer 2 can divide the intensity of the measurement light R3 detected by the measurement photodiode 113 by the intensity of the reference light R2 detected by the reference photodiode 106. Thereby, fluctuations originating from the light source 101 can be offset. Thereby, the SNR can be further improved.

Further, in the first embodiment, the light source light R1 is divided by the polarizing beam splitter 104 into the reference light R2 having an S polarization component and the measurement light R3 having a P polarization component. When viewed from the light source 101, a λ/4 plate 107 is installed after the polarizing beam splitter 104. With this configuration, the measurement light R3 reflected by the mirror 111 finally becomes S-polarized light, and is completely reflected by the polarization beam splitter 104. Thereby, the measurement light R3 reflected by the mirror 111 can be prevented from entering the light source 101, and can be prevented from affecting the light source light R1 emitted from the light source 101.

Note that even though the sample is moving by electrophoresis, the measurement light R3 is irradiated twice at different timings. However, since the speed of light is sufficiently faster than the speed at which the sample moves, it can be considered that the sample is almost stationary with respect to the measurement light R3. Therefore, it is safe to assume that the light transmitted once and the light transmitted twice have exactly the same information on the sample, with the only difference being that the amount of light absorbed by the sample (that is, the amount of attenuation of measurement light R3) is doubled. .

Furthermore, since this embodiment is described on the premise of UV absorption measurement of proteins, the SNR is improved by increasing the light absorption (attenuation) in the capillary 5 due to the reciprocating irradiation of the measurement light R3. On the other hand, in the case of autofluorescence measurement or laser-induced fluorescence measurement, the intensity of fluorescence in the capillary 5 increases due to the reciprocating irradiation of the measurement light R3, so that the SNR can be improved similarly to UV absorption measurement.

[Second embodiment]
FIG. 4 is a schematic diagram showing an example of the overall configuration of a capillary electrophoresis apparatus Ea according to the second embodiment. Note that in FIG. 4, the same components as those shown in FIG.
The capillary electrophoresis device Ea shown in the second embodiment is different from the capillary electrophoresis device shown in FIG. Different from device E. At the end of the injection side 151 of the capillary array 50, each capillary 5 is connected to a separate sample container 3 and an injection side electrode tank 4. In addition, in the detection device 1a, the capillaries 5 are arranged at a constant pitch. Note that the configuration of the analyzer 2 is the same as that of the first embodiment except that the calculation section 211 and the analysis section 212 perform processing on each capillary 5, so the explanation will be omitted.

(Configuration of detection device 1a)
FIG. 5 is a diagram showing a configuration example of a detection device 1a in the second embodiment.
In FIG. 5, the same components as those shown in FIG. 2 are denoted by the same reference numerals, and the explanation thereof will be omitted. Note that in FIG. 5, similarly to FIG. 2, solid line arrows indicate the outward path of light, and broken line arrows indicate the return path of light.
The light source light R1 emitted from the light source 101 is converted into collimated light by the lens 102 which is a collimating lens. The light source light R1 converted into collimated light by the lens 102 passes through the bandpass filter 103 and is coupled by the lenses 121 and 122 to the fiber bundle 130 made up of a plurality of optical fibers 131. Incidentally, the lens 122 is a collimating lens. The lens 121 may be any lens that can condense light. Here, coupling of light to the optical fiber 131 means that light enters the optical fiber 131 and propagates within the optical fiber 131. The configuration of the fiber bundle 130 will be described later. By using such a fiber bundle 130, the light source light R1 can be easily divided.

The lenses 102, 121, and 122 constitute a Koehler illumination system 140, which is an optical system that uniformly illuminates a surface, and plays the role of making the spatial emission intensity distribution of the light source 101 uniform and coupling it to the fiber bundle 130. . Specifically, the lenses 121 and 122 are arranged so that the focal position of the exit side of the lens 121 and the focal position of the entrance side of the lens 122 overlap. With such a configuration, variations in the amount of coupled light between the optical fibers 131 in the fiber bundle 130 are suppressed, and it becomes possible to couple the same amount of light to all the optical fibers 131. Moreover, by making the intensity distribution uniform and coupling, it is possible to suppress fluctuations in the amount of coupled light due to vibrations caused by disturbances in the end face position of the fiber bundle 130, and to prevent a decrease in detection sensitivity.

By configuring the lenses 102, 121, and 122 to form the Koehler illumination system 140 in this manner, uniform light source light R1 can be coupled to each of the optical fibers 131 in the fiber bundle 130. The details of what constitutes the Koehler illumination system 140 will be described later.
Note that the lens 121, the lens 122, and the Koehler illumination system 140 correspond to a light splitting section and a light coupling section. Furthermore, the fiber bundle 130 and the optical fiber 131 correspond to a light splitting section.

The light emitted from each of the optical fibers 131 in the fiber bundle 130 follows the same optical path as in the first embodiment. A brief explanation will be given below.
Each of the source lights R1 emitted from each of the optical fibers 131 in the fiber bundle 130 is converted into collimated light by the lens 123, which is a collimating lens. Each light source light R1 is split into a reference light R2 and a measurement light R3 by a polarizing beam splitter 104, as in the first embodiment. Each reference light R2 is focused by a reference condensing lens 105, and each is detected by a reference photodiode array 124.

On the other hand, each measurement light R3 is converted into circularly polarized light by the λ/4 plate 107, and then focused and irradiated onto each capillary 5 in the capillary array 50 by the lens 108. Each measurement light R3 transmitted through each capillary 5 is converted into collimated light by a lens 109, and then condensed by a lens 110 and irradiated onto a mirror 111. After being reflected by the mirror 111, each measurement light R3 follows the same optical path and is again focused and irradiated onto each capillary 5. Each measurement light R3 transmitted through the capillary 5 is converted into collimated light by the lens 108, and then the polarization state is converted from circularly polarized light to S-polarized light by the λ/4 plate 107. Each of the measurement lights R3 converted into S-polarized light is reflected by the polarization beam splitter 104. Each of the measurement lights R3 reflected by the polarizing beam splitter 104 is focused by a measurement condenser lens 112 and detected by a measurement photodiode array 125. Incidentally, lenses 108 to 110 are collimating lenses as in FIG. 2.

(Configuration of fiber bundle 130)
6A and 6B are examples showing cut surfaces at arbitrary locations of the fiber bundle 130. FIG. FIG. 6A shows the arrangement of the optical fibers 131 when the number of optical fibers 131 is seven, and FIG. 6B shows the arrangement of the optical fibers 131 when the number of optical fibers 131 is eight.
In order to reduce optical energy loss as much as possible, it is desirable that the optical fibers 131 be arranged in a close-packed arrangement as shown in FIG. 6A. In other words, it is desirable that the fiber bundle 130 be configured such that the distance between the plurality of optical fibers 131 is minimized. In the case of the number of optical fibers 131 that cannot be arranged in closest packing, it is desirable to arrange them so that the distance from the center of the fiber bundle 130 to the center of the outermost optical fiber 131 is minimized, as shown in FIG. 6B. . As shown in FIGS. 6A and 6B, the optical fibers 131 on the output side end face of the fiber bundle 130 are arranged in a line. Thereby, the light source light R1 emitted from the light source 101 is divided by the number of optical fibers 131 included in the fiber bundle 130 with substantially uniform intensity.

(Kehla lighting system 140)
Here, the effect of the lenses 102, 121, and 122 in FIG. 5 constituting the Koehler illumination system 140 (verification of uniformity of the amount of coupled light) will be described based on simulation results.
What is important when performing UV absorption measurement by splitting light using the fiber bundle 130 is the uniformity and stability of the amount of light coupled to each of the optical fibers 131 that constitute the fiber bundle 130.
FIG. 7A is a graph of simulation results showing the relationship between the position of the optical fiber 131 in the fiber bundle 130 and the amount of light.
Graph GR11 in FIG. 7A shows a case where an imaging optical system composed of lens 102 and lens 121 is used without providing lens 122 in FIG. 5. Further, graph GR12 shows a case where the lenses 102, 121, and 122 constitute the Koehler illumination system 140 as shown in FIG.

In FIG. 7A, the horizontal axis indicates the position r (μm) of the optical fiber 131 in the fiber bundle 130. Note that the center of the fiber bundle 130 is set to "0" on the horizontal axis of FIG. 7A.
In this simulation, the spatial distribution of the emission intensity of the light source 101 is assumed to be Gaussian with a full width at half maximum of about 0.35 mm. Furthermore, the transmission wavelength of the bandpass filter 103 is 220 nm. Further, the focal length and outer diameter of the lens 102 and the lens 121 are 50 mm and 20 mmφ, respectively. Further, the focal length and outer diameter of the lens 121 are 2.71 mm and 3 mmφ, respectively. The core diameter and NA (Numerical Aperture) of each optical fiber 131 are 105 μm and 0.25, respectively. A simulation is performed based on ray tracing under these conditions.

When an imaging optical system is used, as shown in graph GR11, the spatial distribution of the emission intensity of the light source 101 is reflected, and the amount of coupled light exhibits a Gaussian dependence on the position r of the optical fiber 131. Therefore, the amount of coupled light is larger for the central optical fiber 131 (closer to r=0), and smaller for the peripheral optical fibers 131 (further from r=0). This indicates that the amount of light irradiated to each capillary 511 is different.

On the other hand, when the Koehler illumination system 140 is used, the amount of light coupled to the optical fiber 131 takes a substantially constant value within the range of the position r of the optical fiber 131 of about ±400 μm. Therefore, by arranging the optical fibers 131 within this region (r in the range of about ±400 μm), it becomes possible to couple approximately the same amount of light to all the optical fibers 131. This means that all capillaries 5 are irradiated with substantially the same amount of light (light source light R1). Therefore, by using the Koehler illumination system 140, it is possible to simultaneously measure different samples under the same light amount conditions. Further, the range of the position r of the optical fiber 131 where the amount of coupled light is constant can be freely set by changing the ratio of the focal lengths of the lenses 121 and 122.

Furthermore, regarding the stability of the amount of coupled light, it is necessary to consider the influence of displacement of the optical component (optical fiber 131) due to disturbance. For example, when the position of the input side end face of the optical fiber 131 changes, the amount of light coupled to the optical fiber 131 also changes accordingly. If the amount of light coupled to the optical fiber 131 at the position (x, y, z) of the optical fiber 131 is I, and the amount of positional displacement of the optical fiber 131 is (Δx, Δy, Δz), the amount of coupled light accompanying the displacement of the optical fiber 131 is The amount of variation (hereinafter referred to as variation light amount) ΔI is approximately given by the following equation (11).

Here, ∇ represents spatial differentiation.
From equation (11), it can be seen that the larger the spatial differential ∇I of the coupled light amount of the optical fiber 131, the larger the fluctuating light amount ΔI.

FIG. 7B is a graph showing the absolute value of the fluctuating light amount ΔI that occurs when the position of the incident side end face of the optical fiber 131 fluctuates by Δr in the r direction. Note that FIG. 7B is calculated based on the results of FIG. 7A.

Graph GR21 in FIG. 7B shows the case where an imaging optical system is used. As described above, the combined optical system is a system that is composed of the lens 102 and the lens 121 without the lens 122 shown in FIG. 5. Further, graph GR22 shows a case where the lenses 102, 121, and 122 constitute the Koehler illumination system 140 as shown in FIG. That is, graph GR21 in FIG. 7B is a result based on graph GR11 in FIG. 7A, and graph GR22 in FIG. 7B is a result based on graph GR12 in FIG. 7A.
Further, in FIG. 7B, the horizontal axis indicates the position r (μm) of the optical fiber 131 in the fiber bundle 130. Note that the center of the fiber bundle 130 is set to "0" on the horizontal axis of FIG. 7B.
The magnitude of the positional variation Δr is set to 0.1 μm.

It can be seen that when the imaging optical system is used, the absolute value of the fluctuating light amount ΔI is large in a region other than r=0 μm. This means that the differential of the amount of coupled light with respect to the position r of the optical fiber 131 is large in a region other than r=0 μm. As a result, the detection sensitivity corresponding to the optical fiber 131 arranged in a region other than r=0 μm decreases. That is, in the coupling optical system, the optical fibers 131 arranged other than near the center of the fiber bundle 130 have low robustness against positional fluctuations of the fiber bundle 130.

On the other hand, when the Koehler illumination system 140 is used, as shown in graph GR22, the fluctuating light amount is suppressed in the range of r=-360 to 360 μm due to the effect of uniformizing the intensity distribution (see graph GR12 in FIG. 7A). I understand that. Therefore, by arranging the optical fiber 131 within this region (r=-360 to 360 μm range), the fluctuating amount of light to the optical fiber 131 is suppressed. This makes it possible to prevent a decrease in detection sensitivity. That is, in the Koehler illumination system 140, even if the optical fiber 131 is arranged other than near the center of the fiber bundle 130, the robustness against positional fluctuations of the fiber bundle 130 can be increased.

In the second embodiment, the light source light R1 emitted from the light source 101 is divided into the number of capillaries 5, and is irradiated to each capillary 5 back and forth. As a result, as in the first embodiment, high detection sensitivity can be achieved and multiple samples can be measured simultaneously. As a result, the capillary electrophoresis apparatus Ea according to the second embodiment can achieve higher throughput than conventional capillary electrophoresis apparatuses.

Note that in the second embodiment, the fiber bundle 130 is used to split the light source light R1. However, the present invention is not limited to this, and the same effect can be obtained by dividing the light source light R1 using other means such as a beam splitter or a diffraction grating.

[Third embodiment]
FIG. 8 is a diagram showing a configuration example of a detection device 1b in the third embodiment. In FIG. 8, the same components as those in FIG. 2 are given the same reference numerals, and the description thereof is omitted. The capillary electrophoresis device Ea of the third embodiment is similar to the capillary electrophoresis device Ea shown in FIG. 4 except that the detection device 1a is the detection device 1b, so the capillary electrophoresis device Ea is illustrated and explained. omitted. Note that in FIG. 8, similarly to FIG. 2, solid line arrows indicate the outward path of light, and broken line arrows indicate the return path of light.
In contrast to the second embodiment in which the plurality of capillaries 5 were irradiated with the spatially divided measurement light R3, in the third embodiment one measurement light R3 is temporally divided without dividing the measurement light R3. and irradiates a plurality of capillaries 5. With such a configuration, the detection device 1b according to the third embodiment enables parallel measurement of a plurality of samples.

Comparing the configuration of the detection device 1b shown in FIG. 8 with the configuration of the detection device 1 shown in FIG. 2, they differ in that they include a galvanometer mirror 141 and a plurality of capillaries 5 (5a to 5c). Here, the galvanometer mirror 141 is installed between the λ/4 plate 107 and the lens 108, and changes the optical path between the λ/4 plate 107 and the lens 108.

The angle of the galvanometer mirror 141 is controlled by the analyzer 2b. In this way, by changing the angle of the galvano mirror 141, it is possible to sequentially irradiate each of the capillaries 5a to 5c in the capillary array 50 with the measurement light R3.

(Analyzer 2b)
FIG. 9 is a diagram showing an example of the configuration of an analysis device 2b used in the third embodiment. Note that in FIG. 9, the same components as those in FIG. 3 are denoted by the same reference numerals, and the description thereof will be omitted.
The analyzer 2b shown in FIG. 9 differs from the analyzer 2 shown in FIG. 3 in that it includes a galvanometer mirror control section 213.
The galvano mirror control unit 213 controls the tilt of the galvano mirror 141.

(Control of galvano mirror 141)
FIG. 10 is a graph showing changes in the condensing position of the lens 108 due to control of the galvano mirror 141. Refer to FIG. 8 as appropriate.
The galvanometer mirror control unit 213 of the analyzer 2b sequentially irradiates each capillary 5a to 5c with the measurement light R3 by changing the angle of the galvanometer mirror 141 stepwise with respect to time as shown in FIG. . Here, the focusing position PS1 indicates the position of the capillary 5a, and the focusing position PS2 indicates the position of the capillary 5b. Similarly, the condensing position PS3 indicates the position of the capillary 5c.
Here, the period T1 indicates the time during which the capillary 5a is irradiated with the measurement light R3. Moreover, the period T2 indicates the time during which the capillary 5b is irradiated with the measurement light R3. Moreover, the period T3 indicates the time during which the capillary 5c is irradiated with the measurement light R3.

The time T of one cycle of control of the galvanometer mirror 141 is determined by the sampling frequency of data constituting the electropherogram. The sampling frequency may be set to a value sufficient to obtain a sufficient number of points to construct an electropherogram during one electrophoresis (about 30 minutes), and may be, for example, 2 Hz. In this case, the measurement light R3 needs to be irradiated once to all capillaries 5 during 0.5 seconds, so the period needs to be 0.5 seconds or less. Since the typical response speed of the galvanometer mirror 141 is approximately 10 ms or less, control with a period of 0.5 seconds or less is fully possible. In addition, by using the galvanometer mirror 141 that can be controlled at high speed in this way, unlike Patent Document 1, the time during which the measurement light R3 is irradiated to areas other than the capillary 5 (the space between the capillaries 5 and 5) can be minimized. This makes it possible to suppress a decrease in detection sensitivity.

In the third embodiment, a galvanometer mirror 141 capable of high-speed response is used to divide the measurement light R3 in time and irradiate it back and forth to each of the plurality of capillaries 5. By doing so, high detection sensitivity similar to that of the first embodiment can be achieved, and parallel measurement of a plurality of samples can be achieved. As a result, the capillary electrophoresis apparatus Ea according to the third embodiment can achieve higher throughput than conventional capillary electrophoresis apparatuses. Furthermore, the capillary electrophoresis device Ea according to the third embodiment irradiates each capillary 5 with the measurement light R3 in a time-divided manner, thereby converting signals corresponding to a plurality of capillaries 5 into a single measurement photo. It can be detected by the diode 113. Thereby, it is possible to provide a small and low-cost capillary electrophoresis device Ea.

Note that in the third embodiment, a galvano mirror 141 is used as an optical switching unit that irradiates the capillary 5 with the measurement light R3 divided in time. With such a configuration, it is possible to realize the detection device 1b with excellent high-speed response. However, a method of moving the position of the capillary 5 using a stage or the like may be used as the optical switching unit. It may be configured with a light deflection unit that changes the reflection angle of light, such as a polygon mirror or a MEMS (Micro-Electro-Mechanical Systems) mirror 111. By using such an optical deflection section as an optical switch section, it is possible to realize an optical switching section with better responsiveness than a method in which the capillary 5 is moved using a stage or the like. It is also possible to use a photoacoustic element or the like as an optical switching section.

Note that the present invention is not limited to the embodiments described above, and includes various modifications. For example, the embodiments described above are described in detail to explain the present invention in an easy-to-understand manner, and the present invention is not necessarily limited to having all the configurations described. Furthermore, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Furthermore, it is possible to add, delete, or replace some of the configurations of each embodiment with other configurations.

Further, a part or all of the above-described configurations, functions, units 211 to 213, storage device 202, etc. may be realized in hardware by designing, for example, an integrated circuit. Further, as shown in FIGS. 3 and 9, each of the above-described configurations, functions, etc. may be realized by software by having a processor such as the CPU 201 interpret and execute a program for realizing each function. In addition to storing information such as programs, tables, and files that realize each function on the HD, it can also be stored in memory, storage devices such as SSD (Solid State Drive), IC (Integrated Circuit) cards, and SD (Secure). The data can be stored in a recording medium such as a Digital Versatile Disc (Digital) card or a DVD (Digital Versatile Disc).
Furthermore, in each embodiment, control lines and information lines are shown that are considered necessary for explanation, and not all control lines and information lines are necessarily shown in terms of the product. In reality, almost all configurations can be considered interconnected.

5 Capillary 101 Light source (light source section, irradiation optical section)
102 Lens (irradiation optical section, first collimating lens)
103 Bandpass filter (irradiation optical section)
104 Polarizing beam splitter (irradiation optical section, polarization separation section)
106 Reference photodiode (second photodetector)
107 λ/4 plate (irradiation optical part)
108 Lens (irradiation optical section, second collimating lens)
108,109 Lens (irradiation optical section)
110 Lens (irradiation optical section, third collimating lens)
111 Mirror (reflection part)
113 Measurement photodiode (first photodetector)
121 Lens (light splitting section, optical coupling section)
122 Lens (light splitting section, optical coupling section)
130 Fiber bundle (light splitter)
131 Optical fiber (light splitter)
141 Galvano mirror (optical switching section, optical deflection section)
140 Koehler illumination system (light splitting section, light coupling section)
E, Ea Capillary electrophoresis device R2 Reference light (second polarized component light)
R3 Measuring light (first polarized component light)

Claims (12)

A light source part,
an irradiation optical section that transmits the light emitted from the light source section back and forth through the capillary;
a first photodetection section that detects an optical signal based on the light transmitted back and forth to the capillary;
has
The irradiation optical part is
In order from the light source side,
Polarization separation for transmitting a first polarization component of one of the polarization components of the light emitted from the light source and reflecting a second polarization component of the other polarization component. Department and
a λ/4 plate provided in front of the capillary;
a reflecting section that reflects the first polarized light component that has passed through the capillary toward the capillary;
A capillary electrophoresis device comprising: The irradiation optical section is
In order from the light source side,
a first collimating lens;
a polarization beam splitter as the polarization separation section;
The λ/4 plate,
a second collimating lens provided upstream of the capillary and condensing the light transmitted through the λ/4 plate onto the capillary;
a third collimating lens that is provided downstream of the capillary and upstream of the reflecting section, and focuses the light reflected by the reflecting section onto the capillary;
The capillary electrophoresis device according to claim 1 , characterized in that it has: The irradiation optical part is
A light splitting unit that divides the light emitted from the light source unit into at least the same number of capillaries, and simultaneously irradiates each of the plurality of capillaries with the divided light. Item 1. The capillary electrophoresis device according to item 1 . The light splitting section is
The capillary electrophoresis device according to claim 3 , further comprising an optical coupling section that couples the light emitted from the light source section to each of a plurality of optical fibers. The optical coupling part is
The capillary electrophoresis device according to claim 4 , wherein the spatial intensity distribution of the light emitted from the light source section is made uniform and the light is coupled to each of the optical fibers. The optical fibers constitute an optical fiber bundle,
6. The capillary electrophoresis device of claim 5 , wherein each of the optical fibers in the optical fiber bundle has a close-packed arrangement. The capillary electrophoresis device according to claim 5 , wherein the optical coupling unit is a Koehler illumination system. The irradiation optical part is
The capillary electrophoresis device according to claim 1 , further comprising an optical switching unit that temporally switches the plurality of capillaries that are irradiated with the light. The capillary electrophoresis device according to claim 8 , wherein the optical switching section is an optical deflection section that changes the reflection angle of light. The capillary electrophoresis device according to claim 9 , wherein the light deflection section is a galvanometer mirror. The capillary electrophoresis device according to claim 1 , wherein the first photodetector is a single photodiode that detects the optical signal based on light irradiated onto the capillary twice. The capillary electrophoresis device according to claim 1 , further comprising: a second light detection unit that detects light of the second polarization component among the light from the light source unit that is split by the polarization separation unit. Device.

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